Segmentation of the embryonic vertebrate hindbrain into rhombomeres (r) ensures proper spatial positioning of hindbrain derivatives (e.g. reticulospinal interneurons and motornuclei of the cranial nerves) and is therefore essential for normal neurological activity. This segmentation process is regulated by a growing number of genes, but the function of many of these genes, as well as the regulatory relationships among them, remains unclear. We have identified 12 novel genes expressed in the hindbrain and we hypothesize that these genes act in a regulatory network controlling rhombomere formation. We have developed two aims to test our hypothesis: First we will determine the function of several novel hindbrain genes in rhombomere 4/5 formation. We are focusing particularly on 3-4 genes that we predict are involved in cell sorting at rhombomere boundaries. We will use loss of function approaches (morpholino-mediated knock-down, zinc-finger nuclease-mediated targeted deletions), as well as misexpression approaches (mRNA injections, GAL4:UAS transgenesis), to determine the function of these genes. Second, we will delineate transcription regulatory pathways controlling formation of rhombomere 4/5. Many known r4/r5 genes encode transcription factors, but it is not clear which genes they regulate. We have generated antisera to several of these transcription factors and will use chromatin immunoprecipitation (ChIP) assays to identify direct regulatory relationships among genes acting in r4/r5. We will take both a candidate approach, where we test binding of a specific transcription factor to a predicted target promoter, and a global approach, where we design a hindbrain-promoter tiling-array that will permit identification of all hindbrain promoters bound by a given transcription factor. Our experiments are important because the developing hindbrain is sensitive to disruptions by a variety of factors (e.g. environmental toxins, infectious agents and genetic conditions) that give rise to a range of birth defects - motor control problems such as ataxia, cognitive defects such as autism and craniofacial defects. A better understanding of hindbrain formation will therefore be applicable to a broad set of biological processes and human disease conditions. Our experiments also make novel use of several techniques - ChIP, GAL4:UAS transgenics - in zebrafish.